1. Recombinant Proteins
Methods for producing recombinant proteins are well known in the art; heterologous DNA segments that encode for a particular protein are inserted into host organisms using recombinant DNA technology. By growing the transformant organisms under conditions which induce the expression of proteins, heterologous proteins such as insulin, somatotropins, interleukins, interferons, somatomedins, and the like can be produced. For example, U.S. Pat. Nos. 4,604,359 and 4,332,717 disclose methods for producing human recombinant somatotropin; U.S. Pat. No. 4,431,739 discloses a method for producing recombinant somatotropins; E.P. Patent Application 0 104 920 discloses a method for producing recombinant porcine somatotropin; U.S. Pat. No. 4,443,539 discloses a method for producing recombinant bovine somatotropin; Schoner, Biotechnology, 3(2):151-54, discloses a method for producing recombinant somatotropin, and Buell, Nucleic Acid Res., 13, 1923-38 (1985) discloses a method for producing recombinant somatomedin C.
Typically, the recombinant protein produced has an amino acid sequence which is the same as the amino acid sequence of the native protein.
Often, however, recombinant proteins produced using recombinant DNA techniques have an amino acid sequence which is not the same as the amino acid sequence of the native protein--a modified sequence protein.
It may be desirable to modify the amino acid sequence in a recombinant protein for several reasons. For example, a recombinant protein with a modified amino acid sequence may have physical or chemical properties which make it easier to recover the protein from the fermentation broth, refold and purify the protein during the recovery process, or formulate and administer the protein for the intended purpose. In addition, the modified sequence recombinant protein may have greater bioactivity than the native protein and cause less adverse side effects when administered for its intended purpose.
Also, when administering a modified sequence recombinant protein to an animal for its intended use, it is often difficult to distinguish between the native protein endogenous to the animal and the modified sequence protein administered to the animal. For example, when modified sequence somatotropin is administered to an animal to promote growth, the animal's serum can be assayed for total somatotropin levels using radioimmunoassay (RIA) or other well known techniques. However, it is difficult to differentiate between native and modified sequence somatotropin levels and determine if the modified sequence somatotropin is causing an increase in growth or if the increase in growth is caused by some factor which has increased native somatotropin levels.
Similarly, when somatotropin or any other endogenous protein is being delivered to an animal with a delivery device, it is difficult to determine if the modified sequence protein is being delivered to the animal in the required amounts or if all or part of the the protein can be attributed to endogenous protein.
Methods are, therefore, needed for differentiating between native and modified sequence proteins and for determining the relative amounts of native and modified sequence proteins, particularly modified sequence recombinant proteins, in a sample.
2. Protein Immunology
A macromolecular protein immunogen has several antigenic determinants. Immunizing an animal with a macromolecular protein results in the formation of different antibodies with different specificities for each antigenic determinant; the number of different antibodies depends on the number of antigenic determinants on the macromolecular protein and their inherent immunogenicity.
The immunogenicity of a particular antigenic determinant is dependent upon several factors including the amino acid sequence, conformation, segmental mobility, or hydropathicity of the antigenic determinant.
Antibodies, particularly monoclonal antibodies, formed in response to protein immunogens contain antigen combining sites that are highly specific for individual antigenic determinants on the protein. Thus, an antibody specific for a particular antigenic determinant of a protein will not react with that protein if the antigenic determinant has been deleted, modified or otherwise altered to change its immunogenicity.
When recombinant or synthetic proteins have an amino acid sequence which is not the same as the amino acid sequence of the native protein, the altered amino acids may affect the immunogenicity of an antigenic determinant containing the altered amino acid sequence.
3. Description of References
Pestka, U.S. Pat. No. 4,623,621, discloses the use of antibodies to distinguish monomeric from oligomeric forms of peptides and proteins. The assay employs a single monoclonal antibody in two different assay steps.
Sharp et al, U.S. Pat. No. 4,487,829, discloses the production and use of monoclonal antibodies against adenoviruses.